A major objective Of molecular biology is understanding the mechanisms by which eukaryotic cells regulate gene transcription. Steroid hormone receptors regulate transcription of DNA when bound to their respective hormones, but their mechanism of action is not well understood. The goal of this project is to determine, at the genetic level, the allowed amino acid composition by which the mineralocorticoid receptor (MR) can activate transcription of DNA ' A large panel of mutants of MR gene fragments will be used to test a specific computer model which predicts transcriptional activation regions. An effector plasmid containing an oligonucleotide fragment of the MR gene, based on computer predictions of the region of the receptor most likely to be involved in transcriptional activation, linked to the gene for the DNA binding segment of lac repressor, will be constructed. The ability of the chimeric lac repressor-MR protein to activate transcription will be tested by introducing the effector plasmid, as well as a reporter plasmid, PBR-lac5-tk-CAT, into CV1P monkey kidney cells in tissue culture. The reporter plasmid contains five lac operators, which will serve as DNA binding sites for the chimeric protein, whose synthesis will be directed by the effector plasmid. The attached fragment of MR may then interact with the promoter, and/or other elements which bind at the promoter site, and possibly initiate transcription. This will be assessed by performing assays for CAT enzyme activity on cell extracts. Appropriate controls will be used, and the-stability of the chimeric proteins will be assessed. Various fragments of the MR gene will be tested, until an active piece is found. Mutations of that active oligonucleotide, based on a specific computer model that predicts requirements for transcriptional activation regions, will then be tested in a similar way, in order to determine the amino acid composition essential for activation of transcription. By doing these experiments, we expect to gain better understanding of how steroid hormone receptors regulate gene expression.